Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

TM
GeNei Ouchterlony
Double Diffusion
(for Antigen-Antibody Patterns)
Teaching Kit
Manual

Cat No. New Cat No.

KT70 106208
Revision No.: 00061204
© Bangalore Genei, 2007 © Bangalore Genei, 2007
Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

CONTENTS

Page No.

™ Objective 3

™ Principle 3

™ Kit Description 5

™ Materials Provided 6

™ Procedure 7

™ Observation & Interpretation 9

™ Ordering Information 10

© Bangalore Genei, 2007 © Bangalore Genei, 2007 1

Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

Objective:
To learn the technique of Ouchterlony double diffusion.

Principle:
Immunodiffusion in gels encompasses a variety of
techniques, which are useful for the analysis of antigens and
antibodies. An antigen reacts with a specific antibody to form
an antigen-antibody complex, the composition of which
depends on the nature, concentration and proportion of the
initial reactants.
Immunodiffusion in gels are classified as single diffusion
and double diffusion. In Ouchterlony double diffusion, both
antigen and antibody are allowed to diffuse into the gel. This
assay is frequently used for comparing different antigen
preparations. In this test, different antigen preparations, each
containing single antigenic species are allowed to diffuse
from separate wells against the antiserum. Depending on
the similarity between the antigens, different geometrical
patterns are produced between the antigen and antiserum
wells. The pattern of lines that form can be interpreted to
determine whether the antigens are same or different as
illustrated below:
A B C

Antiserum Antiserum Antiserum

Ag Ag Ag

Fig 1: Different patterns of lines obtained on Ouchterlony

double diffusion.

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Ouchterlony Double Diffusion GeNeiTM
Pattern of Identity: A
The antibodies in the antiserum react with both the
antigens resulting in a smooth line of precipitate. The
antibodies cannot distinguish between the two antigens
i.e., the two antigens are immunologically identical.
(Refer Fig.1: A)
Pattern of Partial Identity: B
In the ‘pattern of partial identity’, the antibodies in the
antiserum react more with one of the antigens (that diffuses
from the left hand well in the figure) than the other. The ‘spur’
is thought to result from the determinants present in one
antigen but lacking in the other antigen. (Refer Fig.1: B)
Pattern of Non-Identity: C
In the ‘pattern of non-identity’, none of the antibodies in
the antiserum react with antigenic determinants that may be
present in both the antigens i.e., the two antigens are
immunologically unrelated as far as that antiserum is
concerned. (Refer Fig.1: C)
© Bangalore Genei, 2007 4 © Bangalore Genei, 2007
Ouchterlony Double Diffusion GeNeiTM
Kit Description:
In this kit, three sets of antigens and antisera are provided.
Each set consists of two antigens and an antiserum against
these antigens. For eg., antiserum A is raised against test
antigen-A1 and test antigen-A2. Students will perform the
ODD assay that will result in three different precipitin lines,
as described in the principle.
KT70 : The kit is designed to carry out 15
Ouchterlony double diffusion experiments.
Perform minimum of 5 experiments at a time.
Duration of experiment: Experiment is carried out over a
span of 2 days, approximate time taken on each day is
indicated below:
Day 1 : 1 hour (Preparation of gel & loading of antigen and
antiserum)
Day 2 : 30 minutes (Observation and Interpretation)
5
Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

Materials Provided: Note:

The list below provides information about the materials • Read the entire procedure before starting the
supplied in the kit. The products should be stored as experiment.
suggested. Use the kit within 6 months of arrival. • Wipe the glass plate with alcohol to make it grease
Quantity free for even spreading of agarose.
Materials KT70 Store • Ensure that the moist chamber has enough wet cotton
(15 expts.) to keep the chamber humid.
Agarose 1.0 g 4°C • Dilute required amount of 10X assay buffer to 1X
concentration (e.g. 1 ml of 10X buffer is mixed with
10X Assay buffer 20 ml 4°C 9 ml of distilled water) before use.
Antiserum (A, B and C)
0.2 ml 4°C • Reconstitute each vial containing the antigen and
(each) antiserum with 0.2 ml of 1X assay buffer. Mix it well
Test antigens 0.2 ml 4°C and allow it to stand for 30 minutes. Store at 4°C. Use
(A1, A2, B1, B2, C1, C2) (each) within 3 months.
Glass plate 5 Nos. 4°C • Assay Buffer: Phosphate buffered saline (PBS).
Gel punch with syringe 1 No. 4°C
Template 2 Nos. 4°C Procedure:
1. Prepare 25 ml of 1.2% agarose (0.3 g /25 ml) in 1X
assay buffer by boiling to dissolve the agarose
Materials Required:
completely.
Equipment : Incubator (37°C). Note: Prepare 5 plates at a time and use on same
Glassware : Conical flask, Measuring cylinder. day.
Reagents : Alcohol, Distilled water. 2. Cool the solution to 55-60°C and pour 4 ml/plate on to
Other Requirements : Micropipettes, Moist chamber 5 grease free glass plates placed on a horizontal surface.
(box with wet cotton), Tips. Allow the gel to set for 30 minutes.
3. Punch wells by keeping the glass plate on the template.

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Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

4. Fill the wells with 10 µl each of the antiserum and the Observation:
corresponding antigens as shown below:
Observe for the presence of precipitin lines between the
antigen and antisera wells. Report the pattern of precipitin
Antiserum A Antiserum B Antiserum C line observed in each case.

Interpretation:
• If pattern A or ‘pattern of identity’ is observed between
the antigens and the antiserum, it indicates that the
Ag A1 Ag A2 Ag B1 Ag B2 Ag C1 Ag C2 antigens are immunologically identical.
• If pattern B or ‘pattern of partial identity’ is observed, it
Fig 2 : Pattern of addition of antigen and antiserum to indicates that the antigens are partially similar or cross-
the wells. reactive.
• If pattern C or ‘pattern of non-identity’ is observed, it
5. Keep the glass plate in a moist chamber overnight at indicates that there is no cross-reaction between the
37°C. antigens. i.e., the two antigens are immunologically
6. After incubation, observe for opaque precipitin lines unrelated.
between the antigen and antisera wells. A B C

Fig 3 : Plate showing pattern of lines obtained following

Ouchterlony double diffusion.

© Bangalore Genei, 2007 8 © Bangalore Genei, 2007 9

Ouchterlony Double Diffusion GeNeiTM Ouchterlony Double Diffusion GeNeiTM

Ordering Information

Product Size Cat #

GeNeiTM Ouchterlony Double

Diffusion (ODD) 1 Pack KT70
Teaching Kit (15 experiments)

Email:
Sales:
geneisales@sanmargroup.com

Customer Support:
geneitechsupport@sanmargroup.com

© Bangalore Genei, 2007 10 © Bangalore Genei, 2007 11